The Construction And Immunogenicity Of Recombinant

The cases of rabies increase greatly in our country in recent years, indicate according to the data that Ministry of Public Health has announced, the death toll of national rabies is 891 in 2001,1122 in 2002,2037 in 2003,2651 in 2004. The rabies is endangering the people's life and health seriously. It is mostly caused by dogs infected by Rabies Virus to bite people. Our country at present use inactivated vaccine and live-attenuated vaccine for dog immunization to prevent rabies, because the cost of the former is expensive, used widely in the developed city mainly; The latter's though relatively cheaper, have potential danger of returning to street strains. Developing the safe , effective , low-priced dog rabies vaccine has important actual meaning.The Rabies Virus is the pathogen of the rabies, it is single negative RNA virus, its genome code N , NS , M , G , L 5 proteins, among them the glycoprotein (G) is the only composition that can stimulate the organism to produce the neutralization antibody to Rabies Virus, and its epitope all centre in the outside membrane district. The living vector vaccine has many advantages such as low cost, effectivity, safety and convenience etc. Canine Adenovirus type 2(CAV-2)attenuated strain is sanctioned to be used in dog infective hepatitis and dog infective throat bronchitis vaccine.CAV-2 has four early replication area, among them E3 district is the non- essential district to replicate, it will not influence the replication of virus to lack or insert the foreign gene of other species. So, CAV-2 has potentiality to develop into live vaccine vector.This research aims at structuring a kind of new-type live vector rabies vaccine through the genetic engineering means, the vaccine regard CAV-2 as vector, add protective antigen gene that is external part of glycoprotein of Rabies virus, when recombinated Canine Adenovirus type 2 infects dogs, recombinated virus rabies virus carry external part gene of glycoprotein of Rabies virus, stimulate the organism to produce the immunity to the rabies virus, thus enable being produced the protection strength to the Rabies Virus by immunization animal. Rabies continues to be a serious problem in developing countries due to the reservoirs of rabies virus in dogs and wildlife vectors. The control of rabies depends on the development of safe, effective, economical vaccines that may be used for preexposure vaccination in animals. For this purpose, the external part of glycoprotein gene of rabies virus strain SRV₉ (RVG) was amplified by the RT-PCR and cloned into pEGFP-Cl with replacement of the GFP gene. The expression cassette pERVG₂ is composed of CMV promoter , external glycoprotein gene and SV40 early mRNA polyadenylation signal. The expression cassette was released by Ase I /Mlu I double digestion and it was cloned rightwards and leftwards into Canine Adenovirus type 2(CAV-2) E3 region plasmid pVAXE3 in which E3 region was deleted partly by Ssp I /DraIII digestion. After the replacement of CAV-2 E3 region in plasmid pCAV-2 which contains CAV-2 genome with the recombinant E3 region, recombinantCAV-2 genome plasmid was obtained. Recombinant CAV-2 genome was released from plasmid by Asc I ICla I digestion,and then transfected into MDCK cells. Two replication-competent recombinant CAV2 expressing Rabies Virus external part of glycoprotein were produced. Vaccination experiment showed that the recombinant viruses can elicit an efficient antibody response in dogs. This research has set up the method of a kind of new, high-efficient method of recombinating Canine Adenovirus type 2,has opened up a new way for the construction recombination Adenovirus live vector vaccine; Developing the live vector rabies vaccine for the first time in nation, the technological means reaches the leading level in the world.